title: Monitoring of dopamine and its metabolites in brain microdialysates: method combining freeze-drying with liquid chromatography-tandem mass spectrometry.
abstract: A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus.
authors.affiliation: Institute of Chemical Technology, Technicka 5, Prague 6, Czech Republic.
authors.forename: Kamila
authors.initials: K
authors.lastname: Syslová
authors.forename: Lukáš
authors.initials: L
authors.lastname: Rambousek
authors.forename: Marek
authors.initials: M
authors.lastname: Kuzma
authors.forename: Věra
authors.initials: V
authors.lastname: Najmanová
authors.forename: Věra
authors.initials: V
authors.lastname: Bubeníková-Valešová
authors.forename: Romana
authors.initials: R
authors.lastname: Slamberová
authors.forename: Petr
authors.initials: P
authors.lastname: Kačer
mesh_terms: D015102:3,4-Dihydroxyphenylacetic Acid / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004298:Dopamine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D004355:Drug Stability; D005260:Female; D005612:Freeze Drying; D006719:Homovanillic Acid / Q000032:analysis* / Q000378:metabolism; D016014:Linear Models; D008694:Methamphetamine / Q000008:administration & dosage; D017551:Microdialysis; D009714:Nucleus Accumbens / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015102:3,4-Dihydroxyphenylacetic Acid; D008694:Methamphetamine; D004298:Dopamine; D006719:Homovanillic Acid